Methods for cell-free dna extraction for non-invasive prenatal screening

Patent metadata

Field	Value
Publication number	US-2023265414-A1
Application number	US-202217977716-A
Country	US
Kind code	A1
Filing date	Oct 31, 2022
Priority date	Oct 26, 2015
Publication date	Aug 24, 2023
Grant date	—

Abstract

Provided herein are methods and systems for cell-free DNA extraction from liquid biological samples. The methods can be employed for determination of fetal DNA fraction and non-invasive prenatal screening of fetal aneuploidies and analyses of other types of cell-free DNA.

First claim

1 .- 31 . (canceled)

32 . A method for improving the yield of cell-free DNA (cfDNA) isolated from a liquid biological sample from subject comprising
a) contacting a liquid biological sample containing cfDNA with a plurality of magnetic beads that reversibly bind to DNA; b) separating the cfDNA-bound magnetic beads from the unbound components of the liquid biological sample; c) transferring the cfDNA-bound magnetic beads to a vessel containing an elution solution; d) separating the magnetic beads from the elution solution by contacting the vessel containing the magnetic beads in the elution solution to a magnetic plate that comprises a ring magnet, and collecting the elution solution containing the cfDNA, wherein the magnetic plate causes the magnetic beads to adhere to the vessel walls.

33 . The method of claim 32 , wherein the magnetic beads comprise cross-linked polystyrene and the surface of the magnetic beads comprises one or more functional groups that reversibly binds to DNA.

34 . The method of claim 32 , wherein the magnetic beads are coated with a silicon-containing coating.

35 . The method of claim 32 , wherein the cfDNA-bound magnetic beads are separated from the unbound components of the liquid biological sample by transferring the cfDNA-bound magnetic beads from the liquid biological sample to another vessel.

36 . The method of claim 32 , wherein the cfDNA-bound magnetic beads are separated from the unbound components of the liquid biological sample by inserting a rod-shaped magnet into the liquid biological sample to magnetically bind the cfDNA-bound magnetic beads, and removing the beads from the unbound components of the liquid biological sample.

37 . The method of claim 36 , wherein the beads are deposited into a vessel containing a wash solution.

38 . The method of claim 37 , wherein the cfDNA-bound magnetic beads are transferred to the elution solution by inserting a rod-shaped magnet into the wash solution containing the cfDNA-bound magnetic beads, and depositing the magnetically-bound beads into the vessel containing the elution solution.

39 . The method of claim 32 , wherein transfer of the magnetic beads is automated.

40 . The method of claim 32 , wherein the liquid biological sample is a plasma sample.

41 . The method of claim 32 , wherein the subject is a pregnant female.

42 . The method of claim 32 , wherein the volume of the liquid biological sample is between about 0.5 mL and about 2 mL.

43 . The method of claim 32 , wherein the volume of the elution solution is less than 200 μl.

44 . The method of claim 32 , wherein step (a) is performed in a multiwell plate.

45 . The method of claim 32 , wherein at least two duplicate liquid biological samples containing cfDNA are contacted with a plurality of magnetic beads.

46 . The method of claim 45 , wherein the cfDNA-bound magnetic beads in each duplicate sample are pooled after step (a).

47 . The method of claim 37 , further comprises transferring the washed cfDNA-bound magnetic beads to two or more wash solutions after step (a).

48 . The method of claim 32 , wherein the magnetic beads are uniform in diameter.

49 . The method of claim 32 , wherein the magnetic beads are about 1 μm in diameter.

50 . The method of claim 32 , wherein the washed cfDNA-bound magnetic beads are incubated in the elution solution for at least 4 minutes.

51 . The method of claim 32 , wherein heat is applied to the elution solution containing the magnetic beads in step (d) to elute the cfDNA.

Assignees

• Quest Diagnostics Invest Llc

Inventors

• Anderson Ben
• Strom Charles
• Tsao David
• Liu Yan
• Sun Weimin

Classifications

• C12N15/1013 Primary

  by using magnetic beads · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q2600/112

  Disease subtyping, staging or classification · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title